Devices and methods for multiplex amplification and detection of DNA using convective heating and label-free microarrays

Abstract

The invention relates to devices and methods for multiplex amplification and detection of DNA using convective heating and label-free microarrays. The present disclosure describes devices and methods for multiplex amplification and detection of nucleic acid targets in a sample. Embodiments of the present disclosure include a mechanical system configured to provide loading, vertical positioning, and clamping of a chip; a thermal control system configured to maintain different temperatures of the chip; and an optical fluorescence imaging system.

Description

[0001] This application is a divisional application of the Chinese patent application filed on April 14, 2021, with application number 201980067896.6 and invention name “Device and method for multiplex amplification and detection of DNA using convection heating and label-free microarray”. The international filing date of the parent application is September 13, 2019, and the international application number is PCT / US2019 / 051057.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 731,495, filed on September 14, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to apparatus and methods for multiplex amplification and detection of DNA using convection heating and label-free microarrays. Background Art

[0005] This invention was made with government support under Grant No. R01CA203964 awarded by the National Institutes of Health and the National Cancer Institute. The government has certain rights in this invention.

[0006] Related technologies

[0007] There is a strong market demand for rapid and multiplexed DNA diagnostics from government agencies, hospitals and physician offices, and civilian consumers. Current commercial qPCR systems are large and expensive and are limited to the simultaneous detection of 4-6 DNA markers of interest (4-6-plex). Two examples of commercial qPCR systems are Luminex-xTag and Biofire-FilmArray, which allow closed-tube 20-plex analysis of a single DNA sample. Both systems require bulky (>20 kg) and expensive (>$50,000) instrumentation and are not suitable for point-of-care applications. Isothermal DNA amplification methods, such as the Alere i influenza test, do not require bulky or complex instrumentation but are limited to 3-plex. Finally, next-generation sequencing (NGS) allows extremely high multiplex analysis of DNA but requires labor-intensive library construction workflows (12 hours or longer) and long sequencing runs (24 hours or longer). These labor-intensive and time-intensive characteristics make NGS unsuitable for point-of-care applications. A comparison of different platforms based on their multiplexing capabilities and instrument affordability / portability is shown in Figure 1 Embodiments associated with the present invention (referred to as the "DonutPCR" platform, discussed in more detail below) uniquely allow for highly multiplexed DNA testing using affordable, portable instrumentation.

[0008] International Patent Application No. PCT / US2017 / 02453 and PCT Patent Publication WO 2017 / 172760 (the '760 Publication) disclose systems and methods developed by the inventors of the pending applications that enable highly multiplexed DNA analysis in a point-of-care environment. With reference to the cyclic nature of the embodiments, the system disclosed in the '760 Publication will be referred to herein as a "DonutPCR system" (or similar terms). The '760 Publication discloses a Donut PCR consumable chip, a method for covalently attaching probes to the inner surface of a fluidic reaction chamber, PCR reagents comprising amplification primers, and detection probe designs. In the '760 Publication, several different instruments were used for PCR amplification, microarray fluorescence imaging, image analysis, and data interpretation. In addition, running the experiment requires many manual intervention steps. Here, a single unified instrument is disclosed that combines all mechanical components, thermal components, optical components, and user interface components to allow a DNA sample-in answer-out platform.

[0009] There appear to be at least three application areas where the high multiplexity and portability of the Donut PCR system uniquely addresses unmet needs: (1) home- and pharmacy-based infectious disease detection and typing, (2) bedside analysis of hospital-acquired infections, and (3) field-based agricultural and veterinary genetic analysis and disease detection applications.

[0010] As previously mentioned, the '760 publication discloses a Donut PCR assay and consumable chip. As used herein, the term "chip" includes a convective fluidic device as described in the '760 publication. Although some of the necessary components of the instrument are described, the '760 publication does not describe a fully integrated Donut PCR instrument in which no manual intervention is required after the initial loading of the chip. Specifically, it does not describe the mechanical components required to load the Donut PCR chip and automatically clamp the chip to the heater to form good thermal contact. In addition, the '760 publication describes the use of a fluorescence microscope, but does not describe the optical elements (filters, lenses, mirrors) required for a separate readout device. Therefore, the present invention is novel and inventive relative to the '760 publication.

[0011] Many quantitative PCR (qPCR) instruments have been invented and commercialized by companies such as Applied Biosystems, Bio-Rad Laboratories, Qiagen, Cepheid, and Roche. These qPCR instruments all have active cooling mechanisms and consume a lot of power. In addition, none of these qPCR instruments use cameras with pixel resolutions less than 100 μm to acquire images of fluorescent spots, which is required to achieve high-reproducibility readouts. Therefore, the present invention is novel and inventive relative to past qPCR instruments.

[0012] Convection PCR has been reported in academic literature in 2002, using two heaters at different temperatures. However, the reported convection PCR instruments did not integrate microarrays for high-multiplex DNA analysis, and therefore, no reported convection PCR instruments used cameras with pixel resolutions less than 100 μm to acquire images of fluorescent spots, which is necessary to achieve high-multiplex readout. Therefore, the present invention is novel and inventive relative to past convection PCR instruments.

[0013] Microarrays utilize spatial separation of specific probes to achieve high readout for DNA analysis. However, commercial microarrays use active fluidics (e.g., pumps) and / or manual washing to remove unbound labeled reagents or amplicons, resulting in an open system that is susceptible to contamination. In contrast, in the present invention, the microarray embedded in the Donut PCR chip is mounted vertically and differentially heated to 95°C and 60°C using two separate heaters. Therefore, the present invention is novel and inventive relative to past microarray technology. Summary of the invention

[0014] In short, the present disclosure provides an apparatus and method for multiplex amplification and detection of nucleic acid targets in biological samples. Embodiments of the present disclosure include: a mechanical system configured to provide loading, vertical positioning and clamping of a chip; a thermal control system configured to maintain different temperatures of the chip; and an optical fluorescence imaging system.

[0015] Certain embodiments include a device for multiplex amplification and detection of nucleic acid targets in a sample, wherein the device includes: a mechanical system configured to provide loading, vertical positioning and clamping of a chip; a thermal control system configured to maintain a first temperature of the chip and a second temperature of the chip, wherein the first temperature is different from the second temperature; an optical fluorescence imaging system configured to collect spatial information in an array of at least 40 pixels by 40 pixels; a power system configured to provide electrical energy to the mechanical system, the thermal control system and the optical fluorescence imaging system; a controller configured to control the operation of the mechanical system, the thermal control system, the power system and the optical imaging system; and a graphical user interface (GUI) configured to allow a user to operate the control system via user interface software.

[0016] In certain embodiments, the chip has a height between 10 mm and 320 mm, a width between 10 mm and 320 mm, and a thickness between 0.5 mm and 10 mm. In some embodiments, the GUI is a touch screen interface. In certain embodiments, the touch screen interface is integrated into a smart phone. In certain embodiments, the mechanical system includes a chip holder, a frame, a sliding member, and a support member. In certain embodiments, the chip holder includes a slot, a locating slot, and a motion control kit. In certain embodiments, the frame includes a base, a main structural carrier, a pressure rod locator, a pressure rod, and a motion control kit. In certain embodiments, the motion control kit includes a motorized moving member and a holder thereof.

[0017] In some embodiments, the motorized moving component includes a linear actuator or a stepper motor. In specific embodiments, the sliding component includes a sliding platform and a sliding rod. In some embodiments, the support component includes a track bracket. In specific embodiments, the thermal control system includes a plurality of temperature sensors, a plurality of heating blocks, and a heat source. In certain embodiments, the plurality of temperature sensors include a resistance temperature detector (RTD), a thermistor, a thermocouple, or an IR sensor. In specific embodiments, one of the plurality of temperature sensors is embedded in the heating block. In some embodiments, one of the plurality of temperature sensors is coupled to the surface of a heating block in the plurality of heating blocks. In specific embodiments, the plurality of heating blocks comprise aluminum, stainless steel, or brass.

[0018] In certain embodiments, the plurality of heating blocks contact at least 50% of the total surface area of ​​the chamber in common. In certain embodiments, the heat source comprises a bonded flexible heater, a thermal probe, or a heating wire. In certain embodiments, the fluorescence imaging system comprises a light source, an optical module, and a detector having at least 40×40 pixels. In certain embodiments, the detector is a camera. In certain embodiments, the camera comprises a scientific-grade camera or a smart phone camera. In certain embodiments, the light source comprises an arc lamp, a vapor lamp, a light-emitting diode (LED), or a laser. In certain embodiments, the optical module comprises an excitation filter, a dichroic mirror, a beam splitter, an emission filter, a plane mirror, an objective lens, and / or an optical lens. In certain embodiments, the mechanical system comprises one or more plastics. In certain embodiments, the mechanical system comprises polylactic acid (PLA), polycarbonate (PC), acetonitrile butadiene styrene (ABS), or ceramics. In certain embodiments, the power system comprises an AC / DC power supply, a MOSFET, a switch, an amplifier, a diode, a transistor, and a resistor. In certain embodiments, the controller comprises a microcontroller and / or a PID controller. In certain embodiments, the microcontroller comprises a Raspberry Pi, an Arduino, or a Genuino.

[0019] Certain embodiments include a method for analyzing a sample in a chip using an apparatus as described herein, including, for example, an apparatus according to claim 1. In a specific embodiment, the method includes: loading a chip into the apparatus; operating a mechanical system to clamp the chip between a first heating block and a second heating block; heating the second heating block to a second temperature, wherein the second temperature is different from the first temperature; directing excitation light from a light source to a surface of the chip; detecting emission light from the chip; and analyzing the emission light from the chip.

[0020] Some embodiments further include generating a data report that records the analysis of the light emitted from the chip. Specific embodiments further include releasing the chip from between the first heating block and the second heating block. Certain embodiments further include unloading the chip from the device. In specific embodiments, loading the chip includes operating a motor to perform a linear movement. In some embodiments, loading the chip includes operating a mechanism configured to pull the chip into the device. In specific embodiments, clamping the chip includes operating a mechanism using a self-locking motor, a cam follower combination, or a spring.

[0021] In some embodiments, during operation, the first temperature is maintained between 75°C and 105°C. In a specific embodiment, during operation, the second temperature is maintained between 30°C and 75°C. In some embodiments, the first temperature and the second temperature are controlled by a microcontroller program that changes the heat source power based on feedback from a temperature sensor. In a specific embodiment, the first temperature and the second temperature are controlled by a proportional-integral-differential (PID) controller. In some embodiments, the first temperature sensor is embedded in the first heating block. In a specific embodiment, the second temperature sensor is embedded in the second heating block. In a specific embodiment, the first temperature sensor is coupled to the surface of the first heating block. In some embodiments, the second temperature sensor is coupled to the surface of the second heating block. In a specific embodiment, the excitation light source forms an angle between 30° and 90° with the surface of the chip. In some embodiments, detecting the emitted light from the chip includes operating a camera to continuously acquire images at a frequency of not less than 1 image every 2 minutes.

[0022] Any embodiment of any of the methods, compositions, kits, and systems of the invention may consist of or consist essentially of the described steps and / or features, rather than comprise / include / contain / have the described steps and / or features. Thus, in any claim, the term "consisting of" or "consisting essentially of" may replace any of the above open-ended linking verbs to change the scope of a given claim from the scope using the open-ended linking verb to another scope.

[0023] Unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, use of the term "or" in the claims means "and / or," although the present disclosure supports a definition involving only alternatives and "and / or."

[0024]

[0043] The term "about" is used throughout this application to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0025] Under long-standing patent law, "a" or "an" when used in conjunction with "comprising" in a claim or specification means one or more unless otherwise noted.

[0026] According to the following detailed description, other objects, features and advantages of the present invention will become apparent. However, it should be understood that although the preferred embodiment of the present invention is indicated, the detailed description and specific embodiments are given by way of illustration only, because through this detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. The patent or application file may contain at least one color drawing. Upon request and payment of the necessary fee, the Patent Office will provide copies of this patent or patent application publication with one or more color drawings.

[0028] Figure 1 A graphical comparison of the multiprocessing capabilities of different platforms as well as instrument affordability and portability.

[0029] Figure 2(a) to Figure 2(e) Convective flow in a capillary and flow in a chip, along with associated manufacturing and operating principles, are shown in accordance with the present disclosure.

[0030] Figure 3 A workflow diagram for device operation according to the present disclosure is shown.

[0031] Figure 4(a) to Figure 4(c) A partially exploded view of a device according to the present disclosure is shown, along with photographs of a prototype device and imaging results from the prototype.

[0032] Figure 5 A conceptual functional flow chart of an apparatus according to the present disclosure is shown.

[0033] Figure 6(a) to Figure 6(d) A perspective view and a photographic representation of a chip holder and a chip are shown in accordance with an apparatus of the present disclosure.

[0034] Figure 7(a) to Figure 7(c) Schematic diagrams of alternative chip holders, loading mechanisms, and clamping mechanisms of an apparatus according to the present disclosure are shown.

[0035] Figure 8(a) to Figure 8(c) A perspective view of the various sub-frames and the assembled frame of an apparatus according to the present disclosure is shown.

[0036] 9(a) and 9(b) show perspective views of a strut and assembled frame of an apparatus according to the present disclosure.

[0037] Figure 10(a) to Figure 10(d) The mechanical system of a device according to the present disclosure is shown in an unclamped position and a clamped position.

[0038] 11( a ) and 11 ( b ) show orthographic and perspective views of a heating block alone and in an assembled frame of an apparatus according to the present disclosure.

[0039] Figure 12(a) to Figure 12(g)Orthographic, perspective, and photographic representations of loaded thermal control system components, as well as response curves of the heating block temperature, are shown in accordance with the present disclosure.

[0040] 13( a ) and 13 ( b ) show schematic diagrams of a fluorescence imaging system according to an apparatus of the present disclosure. DETAILED DESCRIPTION

[0041] Exemplary embodiments of the present disclosure include three major components: (1) Chip: a toroidal reaction chamber for reliable convective PCR without active cooling or fluidics; (2) Assay and Readout: using a single fluorescent channel, 50 or more DNA targets can be simultaneously detected and analyzed in a closed-tube manner via spatial separation of probes in a microarray; and (3) Device: a portable and affordable instrument that enables multiplex amplification and real-time readout.

[0042] The principle of convection PCR is that aqueous solutions can be controlled to circulate due to temperature-induced density differences (Rayleigh-Bénard convection). In short, hotter solutions are less dense, while cooler solutions are more dense, so gravity can drive the circulation of the differentially heated solutions. Since no active cooling or fluidics are required, the size and weight of a convection PCR instrument will be significantly lower than conventional PCR using a Peltier system.

[0043] Convection PCR was first conceptually introduced and experimentally demonstrated in 2002 using a vertical capillary as a reaction chamber, as shown in FIG2(a). However, the reaction chamber has a "dead zone" with low circulation speed, resulting in nonspecific DNA amplification and primer dimer formation. These relative disadvantages of convection PCR using capillaries make convection PCR an unpopular alternative to conventional PCR, and therefore its commercial use is low. In contrast, the engineered annular reaction chamber shown in FIG2(b) eliminates the dead zone in the chip and promotes uniform circulation of the PCR solution through the 95°C temperature zone and the 60°C temperature zone in the chamber. The Donut PCR fluidic chip 100 shown in FIG2(b) is designed to achieve more uniform temperature control than a capillary so that convection PCR can perform diagnostic-grade DNA analysis. The PCR solution is injected into the reaction chamber via an inlet orifice, while another orifice allows air to bypass. The inner circular insert in the middle ("island") prevents the occurrence of a dead zone where the PCR mixture stays at an uncontrolled temperature for a long time. The manufacturing and assembly process of the Donut PCR chip 100 is shown in FIG2(c). An overview of the label-free microarray technology that can be used in conjunction with the Donut PCR chip 100 is shown in FIG2(d). The amplicons and dNTPs are unlabeled, and local fluorescence enhancement is achieved by replacing the quencher-labeled oligonucleotides with the unlabeled amplicons. Therefore, the technology avoids the open tube cleaning step. FIG2(e) provides fluorescent images of the label-free microarray before (upper figure) and after (lower figure) hybridization with the amplicon mixture. The images here were taken using a Zeiss Axio Observer fluorescence microscope.

[0044] Conventional quantitative PCR (qPCR) instruments use different spectral wavelengths to achieve multiplex analysis of different DNA targets. However, the number of non-overlapping visible wavelength fluorophores is limited to 5-6. For applications ranging from infectious disease and antibiotic resistance analysis to agricultural genetic analysis, more than twenty different targets must usually be detected. Sample reduction can be performed, for example, by analyzing twenty different DNA targets in a sample via four reactions, with five targets tested per reaction. However, in practice, this is cumbersome and sacrifices sensitivity when samples are limited. Microarrays use spatial separation to achieve highly multiplexed processing using a single fluorescent wavelength, but require labor-intensive and open-tube washing steps to suppress fluorescent background. Complex microarray workflows make traditional microarrays unsuitable for in vitro diagnostic (IVD) use. In contrast, microarrays printed on the inner surface of a circular Donut PCR chip are label-free microarrays and do not require washing to remove excess amplicons or reaction reagents.

[0045] In order to perform multiple amplification and detection of many DNA targets in a Donut PCR chip, the chip needs to be lightly mounted on two heaters at different temperatures to form good thermal contact. To prevent excessive background light, the Donut PCR chip needs to be moved to a dark area, or the chip loading door must be sealed. The Donut PCR chip also needs to be illuminated with light of appropriate wavelength, intensity, and focus by using properly selected and positioned filters and optical elements. The camera takes pictures of the microarray at fixed time intervals, and then the image is interpreted using software to determine the presence / absence of bright spots. An exemplary embodiment of the present disclosure provides an integrated device for implementing each of the above processes.

[0046] Reference now Figure 3 , provides a schematic diagram of an apparatus 200 for multiplexed amplification and detection of nucleic acid targets in a sample, and an overview of a workflow diagram for use with the apparatus 200. Figure 3 As shown, the sample 110 can be mixed with one or more PCR reagents and then loaded into the chip 100 via a pipette. Then, the chip 100 (with the sample 110) can be sealed and inserted into the device 200 for analysis. The user can control the operation of the device 200 via a graphical user interface (GUI) 210. In certain embodiments, the GUI 210 can be wirelessly connected to the device 200, and in certain embodiments, the GUI 210 can be integrated into a mobile device 220 (including, for example, a smart phone, a laptop computer, a tablet computer, or other suitable device). The GUI 210 can be used to initiate analysis of the sample 110 via the device 200 and display image results. The device 220 can be used to send the test results to a doctor, a nurse, or other healthcare professional.

[0047] Reference now Figure 4(a) to Figure 4(c) , providing an overview of the device 200. FIG. 4( a) shows a three-dimensional view of the assembled components (excluding the housing). FIG. 4( b) provides a photograph of an engineering prototype of the device 200 (again, excluding the housing). FIG. 4( c) shows a fluorescent image of a 100-point array in a Doughnut PCR chip 100 imaged by an embodiment of the device 200 shown in FIG. 4( b). Certain embodiments of the device 200 may include three-dimensionally printed structural components (e.g., using a suitable device such as an Ultimaker 3). Other components may include a large number of off-the-shelf modules. In the embodiment shown in FIG. 4( b), a smart phone is used as a camera, image processing unit, user interface, and wireless data transmitter. Figure 4(a) to Figure 4(c) The embodiment shown is wall powered, but also has an internal mobile power supply that is intended to power up to 60 minutes of operation (e.g., a complete sample analysis plus any additional time margin that may be required).

[0048] Figure 5 A conceptual diagram or flow chart of the analysis performed by the apparatus 200 is provided, which includes steps performed in four major systems: fluorescence imaging 210, user interface 220, mechanics 230, and thermal control 240. An overview of the general process for operating the apparatus 200 is provided below in conjunction with the Doughnut PCR chip 100 loaded with appropriate samples, dNTPs, DNA polymerase, and buffers.

[0049] The user can initially turn on the power of the device 200, so a system check is performed before initialization (verifying controller connections, checking camera functions and light source illumination, etc.). During the initialization of the device 200, the thermal control system is turned on, and then the heating blocks are activated to a predetermined temperature and maintained at a stable temperature. More detailed information on the temperature control method will be further explained in the thermal control system section. With the heating block process, temperature recording can also be started at the same time. The thermal control system can run continuously until the experiment is completed. The system initialization process also includes turning on the fluorescence imaging system, checking the light source energy, confirming the camera focus, and re-ensuring that the initialization image is ready to be taken for recording. Next, the system resets the position of the moving mechanical parts. For example, this may include moving back a frame that fixes the heating block to an unclamped position, setting the chip holder to an unloading / ejection position, and preparing to load the chip.

[0050] The device 200 may include a display indicating that the device is ready for an experiment or analysis. The user may then prepare to load the chip into the device 200. Next, the user may insert and load the chip, ensuring that the chip has been loaded and placed. The device 200 may be configured so that the heating block will then clamp the chip. The device 200 then indicates that it is ready to start and displays a countdown for the experiment / analysis. The device 200 then starts / resets the fluorescence imaging system timer, using a camera to take an image at predefined time intervals (e.g., every 45 seconds). After each predefined time interval, the light source will turn on to excite the fluorophores in the chip. In some embodiments, a laser or LED light with an excitation filter may be considered as a potential light source.

[0051] The device 200 can then use a camera to capture and save a fluorescent image of the current chip. Different types of fluorophores and instrument designs will require different fluorescent filter sets. Next, the light source power can be turned off and the next image can be taken at a predefined time interval. This can be repeated until the experiment / analysis is completed. After the experiment is completed, the thermal control system and the fluorescent imaging system will be turned off. After the analysis is completed, the chip will be automatically ejected. Due to the high temperature of the chip, the user should use caution. The heating block in the mechanical system can then be released and the chip can be ejected. The device 200 can continue to analyze the image and finalize the data report. The user can then continue to the next experiment or turn off the power device 200.

[0052] Reference now Figure 6(a) to Figure 6(d) , FIG6(a) shows a three-dimensional view of a chip holder 205 configured to hold one or more chips 100. FIG6(b) shows the position of a chip 100 after being loaded into the holder 205, while FIG6(c) shows the chip 100 in an unloaded position (left) and a loaded position (right). FIG6(d) provides a photograph of a chip holder 205 (fabricated via three-dimensional printing in this example) loaded with chips 100.

[0053] Apparatus 200 includes components in a mechanical system that will perform functions including, but not limited to, chip loading and heat block clamping.

[0054] The function of the chip holder 205 in the device 200 is to insert the doughnut PCR chip 100 and guide the chip to move vertically to a predetermined position. The chip holder includes three main parts: a slot 206, a positioning slot 207, and a motion control kit 208. The slot 206 is extruded from the surface of the instrument for chip insertion. The chip 100 is inserted into the slot 206, which will support the chip 100 to move vertically up and down, and accompanied by the guidance of the motion control kit 208, the chip 100 is then accurately positioned in the positioning slot 207.

[0055] The two vertical bars beside the positioning groove 207 serve as guide rails 209 and guide the chip 100 to move vertically up and down. The motion control kit 208 includes two parts, a moving part 211 and a fixing device 213 thereof. In some embodiments, the moving part 211 utilizes a linear motion motor, such as a linear actuator or a stepper motor. The fixing device 213 of the motion control kit 208 is used to fix and ensure the stability of the moving part 211 during the movement of the chip 100. The slot 206, the positioning groove 207, the guide rails 209 and the fixing device 213 together form an integrated chip holder 205. In an exemplary embodiment, the moving part 211 of the motion control kit 208 is assembled on the chip holder 205. After the entire chip holder 205 and the moving part 211 are assembled, they will be fixed inside the device 200. The vertical movement process of the chip 100 is guided and indicated by the motion control kit 208, wherein the doughnut PCR chip 100 is an object that moves vertically.

[0056] Before the experiment, the chip holder 205 is reset to the unloading position, and the moving part 211 (e.g., linear actuator) will be extended as shown in the left view of Figure 6 (c). After the chip 100 is inserted, the chip will move vertically downward together with the shortened linear actuator until the chip 100 is accurately positioned in the positioning groove 207. At that time, both the upper and lower edges of the chip 100 will be assembled in the slot 206 (a part of the lower edge of the chip 100) and the positioning groove 207 and remain vertical, as shown in the right view of Figure 6 (d) and Figure 6 (c). After the experiment, the moving part 211 will extend and guide the chip 100 to move upward so as to eject the chip 100.

[0057] In other embodiments (not shown), the chip holder may include different shapes to accommodate different types of equipment. In certain embodiments, the chip holder may be moved vertically up and down, rather than just moving the chip itself.

[0058] Before performing an experiment or analysis, the chip holder 205 will be driven by the moving part 211, and this will push the socket 206 out of the device 200 for chip insertion. After the chip 100 is inserted, the chip 100 will directly contact the bottom of the positioning groove 207, and the upper edge of the chip will be parallel to the socket 206. Then, the moving part 211 will drive the entire chip holder 205 to move downward, and then pull a part of the socket 206 back to the device 200, and the chip loading process is completed.

[0059] After the experiment, the moving part 211 will move upward together with the chip holder 205 carrying the chip 100 to squeeze the socket 206 out of the surface of the instrument 200, and then the chip 100 can be removed.

[0060] FIG. 7( a) illustrates an alternative design for a chip holder 205 configured to hold a chip 100. FIG. 7( b) provides four different configurations of a loading mechanism 215 configured to load a chip holder 205. Each embodiment includes a heater, but includes different components for moving the chip holder 205 into and out of a position between the heaters. The first embodiment includes a stepper motor having a lead screw and a lead nut. The second embodiment includes a stepper motor that rotates a cam about a horizontal axis to raise and lower a follower coupled to the chip holder. The third embodiment includes a stepper motor that rotates a cam about a vertical axis to raise and lower a follower coupled to the chip holder. The fourth embodiment includes a motor coupled to a roller gear and a roller configured to engage and raise and lower the chip holder 205. FIG. 7( c) illustrates three different embodiments of a clamping mechanism 225 configured to clamp a heater on each side of a chip holder and a chip. Each embodiment includes a stepper motor configured to control the movement of a component, and a clamping block configured to contact the heater. The first embodiment includes a cam coupled to a stepper motor and a follower coupled to a clamping block. This embodiment also includes a plurality of springs on the guide rail, wherein the springs are biased to apply a force to the clamping block toward the heater (e.g., the springs are biased toward a clamped position). When the cam rotates, it can engage the follower and counteract the spring force to guide the clamping block to a position away from the heater (e.g., to an unclamped position).

[0061] The second embodiment in FIG. 7( c) shows a clamping mechanism 225 having a similar configuration to the previous embodiments. However, instead of a cam, this embodiment utilizes a lead screw and lead nut arrangement to move the clamping block toward and away from the heater. The third embodiment shown in FIG. 7( c) shows a clamping mechanism 225 having a stepper motor and cams that engage a clamping block that pivots about a hinge mechanism. This embodiment also includes a torsion spring configured to bias the pivoting clamping block toward the heater.

[0062] Reference now Figure 8(a) to Figure 8(c) 9(a) and 9(b), showing perspective views of the frames alone and assembled. The main function of the frame is to hold the heating block vertically. The frame shown here has two parts, sometimes referred to herein as "frame Alpha" and "frame Beta".

[0063] FIG8( a) provides a perspective view of sub-frame 410 (also referred to as frame Alpha), while FIG8( b) provides a perspective view of sub-frame 420 (also referred to as frame Beta). FIG8( c) shows sub-frame 420 positioned proximal to sub-frame 410 to form frame 450. Both frames include three main components: a base, a main structural carrier, and a strut. The base is used for connection and fixation between the overall frame and the sliding platform. FIG8( a) shows sub-frame 410 having a base 411 and a main structural carrier 412, while FIG8( b) shows sub-frame 420 having a base 421 and a main structural carrier 422. FIG9( a) shows struts 413 and 414 used in conjunction with sub-frame 410, and struts 423 and 424 used in conjunction with sub-frame 420. In addition, Figure 9(a) shows heating blocks 415 and 416 coupled to compression bars 413 and 414, and heating blocks 425 and 426 coupled to compression bars 423 and 424. Figure 9(b) shows the frame 450 assembled with subcomponents. For clarity, not all individual components are labeled in Figure 9(b).

[0064] The main structural carriers 412 and 422 play a major role in the frame 450, and their main function is to fix the pressure rods 413, 414, 423 and 424 on the clamps 417 and 427 (marked in Figures 8(a) and 8(b)). In some embodiments, during manufacturing, the bases 411 and 421 and the structural carriers 412 and 422 can be made into single parts respectively. Each component can be made of a hard material so as to maintain its shape and form. In the three-dimensional printing prototype, polylactic acid (PLA) material and acrylonitrile butadiene styrene (ABS) material, etc. can be suitable materials.

[0065] The function of the pressure rods 413, 414, 423 and 424 is to fix the heating blocks 415, 416, 425 and 426. As shown in Fig. 9 (a) and Fig. 9 (b), the shape of the pressure rods 413, 414, 423 and 424 is a flat U-shape. The pressure rods need to maintain their shape at high temperatures, and it is recommended to use heat-resistant materials. In some embodiments, polycarbonate (PC) or ceramic materials can be used for the pressure rods.

[0066] After the compression rods 413, 414, 423, and 424 are coupled to the frame 450 (via the sub-frames 410 and 420), the heating blocks 415, 416, 425, and 426 will be placed vertically. The sub-frame 410 will assemble the first two heating blocks 415 and 416, and the sub-frame 420 will assemble the second two heating blocks 425 and 426. The configuration (e.g., shape and size) of the sub-frames 410 and 420 (also referred to as the frame Alpha and the frame Beta) are different to facilitate the convenience of two fully assembled sub-frames. Figure 8(a) to Figure 8(c)As shown, the linear actuator 430 is coupled between the sub-frame 410 and the sub-frame 420. When the linear actuator 430 is extended, the heating blocks 425 and 426 will move closer to the heating blocks 415 and 416 until the heating blocks fully engage or clamp the chip 100. When the linear actuator 430 is shortened, the heating blocks 425 and 426 will move away from the heating blocks 415 and 416 until the heating blocks successfully separate or release the chip 100.

[0067] Figure 10(a) to Figure 10(d) An overview of a mechanical system 500 configured to provide vertical loading and clamping of a chip 100 is provided. FIG10( a ) shows a side view of the mechanical system 500 in an unclamped position. In this view, the linear actuator 430 is in a retracted position and the subframe 420 is moved toward the linear actuator 430.

[0068] 10( b ) shows a side view of the mechanical system 500 in a clamped position. In this view, the linear actuator 430 is in an extended position, and the subframe 420 is moved away from the linear actuator 430 and toward the chip 100 .

[0069] FIG. 10( c ) provides a perspective view of the mechanical system 500 in a clamped position, while FIG. 10( d ) provides a photograph of the mechanical system 500 with a loaded Doughnut PCR chip 100. For clarity, the mechanical system 500 is not shown. Figure 10(a) to Figure 10(d) All components in the drawings are marked with reference numerals.

[0070] The subframes 410 and 420 may be supported by a sliding member 460 to facilitate forward and backward movement of the frame. In a specific embodiment, a commercially available linear bearing platform (identified by part number SC8UU) may be used for the sliding member. In certain embodiments, the sliding member may include a sliding platform, a sliding rod, and a clamping bracket. The surface of the sliding platform is horizontal and is coupled to the frame bases 411 and 421. The sliding platform is coupled to the sliding rod and can move back and forth along the sliding rod.

[0071] In an exemplary embodiment, the sliding platform design can be customized as long as it maintains its function to minimize friction. In one embodiment, the sliding rod is a stainless steel surface polished cylinder with a diameter of 8 mm. In some embodiments, two sliding rods can be used for the platform. The sliding rod can be selected from many commercially available products and can be any of a variety of sizes and materials. The clamping bracket is used to fix and position the sliding rod. In some embodiments, three sets of clamping brackets can be used, each set containing two clamping brackets. One set of clamping brackets forms a partition and buffer between two sets of sliding platforms to prevent excessive movement, thereby avoiding damage to the Donut PCR chip 100 during the clamping process.

[0072] An exemplary embodiment of the present invention also includes a thermal control system configured to maintain two different temperatures of the chip 100 via heating blocks 415, 416, 425 and 426. Now referring to Figure 11(a), front and rear views of heating blocks 415 and 416 are provided. As shown in Figures 11(a) and 11(b), the heating block 416 includes a wedge-shaped slit 418 near one edge of the block 416. As further described below, the wedge-shaped slit 418 allows fluorescent imaging. When the heating block 416 is in a clamped position, the center of the wedge-shaped slit 418 corresponds to the position of the unlabeled microarray on the loaded Doughnut PCR chip 100. In the illustrated embodiment, the heating blocks 415 and 425 are set to 95°C during operation, and the heating blocks 416 and 426 are set to 60°C during operation.

[0073] Figure 12(a) to Figure 12(g) Various aspects of an exemplary embodiment of a thermal control system according to the present invention are shown. FIG. 12( a ) shows a schematic perspective view of a three-dimensional view of a heat source 431 attached to a heating block, while FIG. 12( b ) provides a photograph of a heat source 431 configured as a bonded flexible heater coupled to a heater block (e.g., block 426). FIG. 12( c ) shows a partial cross-sectional view of a temperature sensor 432 embedded in heating blocks 425 and 426 beneath a Doughnut PCR chip chamber.

[0074] FIG12( d) shows a temperature sensor 432 attached to the chip 100 and located on the surface of the heating blocks 425 and 426. FIG12( e) provides a photograph of the temperature sensor 432 configured as a thermocouple and a thermistor. In addition, FIG12( e) shows various temperature sensors, including a surface-bonded RTD sensor. FIG12( f) provides a photograph of the thermal control system 600, including a prototype of the thermal control board 610. FIG12( g) shows a response curve of the heating block temperature measured in degrees Celsius relative to time in seconds.

[0075] In certain exemplary embodiments, the heating block is a heat conductive plate with a polished surface. In a specific embodiment, the heating block should exhibit a width and length that can cover at least half of the Donut PCR chip. The principle of the heating block is to heat the chip to a predetermined temperature by clamping the chip to the polished surface of the heated heating block. In the specific embodiment shown in the drawings, a total of four separate heating blocks are used. During operation of the instrument, two of the heating blocks maintain a temperature of 95°C and the other two maintain a temperature of 60°C.

[0076] The total number of heating blocks used for stable temperature control may vary. For example, in Figures 11(a) and 11(b), it is sufficient to use only heaters 415 and 416, considering the good feedback control loop for maintaining the temperature. In the embodiment shown in the accompanying drawings, four heating blocks are used to achieve temperature robustness and fault tolerance. The material used in the heating blocks can be aluminum, stainless steel or brass or other suitable thermally conductive materials. The thickness of the heating blocks can be adjusted based on the desired temperature and the properties of the heat source. In the exemplary embodiment shown in the accompanying drawings, a three millimeter thick aluminum plate is used for the heating blocks.

[0077] In an exemplary embodiment, the temperature sensor may be assembled on the surface of the heating block or embedded in the heating block, as shown in FIG. 12( c) and FIG. 12( d). In the illustrated embodiment, the temperature sensor is embedded in the heating block at the position where the heating block contacts the Donut PCR chip so as to obtain the most accurate temperature reading of the temperature of the Donut PCR chip (see FIG. 12( c)). In an exemplary embodiment, the temperature sensor should be able to accurately measure temperatures from 20°C to 105°C, with a response time of ≤1 second and an accuracy of ≤1.5°C. The embodiment shown in the accompanying drawings uses an OMEGA T-type thermocouple. Experimentally, it has been observed that other types of thermocouples can achieve similar results. Alternatively, a resistance temperature detector (RTD) or a thermistor may be used instead. Note that if an embedded temperature sensor strategy is adopted, the shape of the heating block should adapt to the shape of the temperature sensor.

[0078] Exemplary embodiments of the present disclosure also include a heat source that provides heat to the heating block. The illustrated embodiment includes a Kapton polyimide bonded flexible heater. The heater adheres to the surface of the heating block but does not directly contact the Donut PCR chip. The heater maintains temperature by varying the power supplied to the heat source. Alternatively, any heating plate containing a heating wire may replace the heating block or heater. Specifically, it should be noted that the heat source can quickly heat the heating block by converting electrical energy into heat, but cannot provide rapid cooling.

[0079] The exemplary embodiment further includes a temperature sensor feedback loop as a central aspect of the thermal controller. The thermal controller reads the temperature from the temperature sensor and provides guidance on the power to the heating block to (1) quickly achieve heating to the desired temperature, and (2) accurately maintain the temperature even if there may be fluctuations due to the environment. The exemplary embodiment can use an Arduino microcontroller through an amplifier module to accurately read the temperature of the thermocouple and control the power of the heat source. Alternatively, a general PID (proportional-integral-derivative) controller can also achieve the same functionality.

[0080] Exemplary embodiments of the present invention also include an optical fluorescence imaging system configured to collect spatial information in an array of at least 40 pixels by 40 pixels. Referring now to FIG. 13( a) and FIG. 13( b), an overview of an exemplary optical fluorescence imaging system is shown. FIG. 13( a) shows a system 700 including a conventional or typical fluorescence microscope arrangement, while FIG. 13( b) shows a schematic diagram of an optical system 800 for a prototype instrument.

[0081] Fluorescence imaging of the Donut PCR chip 100 requires: a light source 710; an optical module 720 for directing and focusing light to an appropriate area of ​​the chip 100; an emission filter 730 for reducing background signals of other wavelengths; and a photodetector array 740 or a camera for image acquisition.

[0082] In the embodiment shown in FIG. 13( a), the system 700 further includes a dichroic mirror 750, which is a high-pass mirror that will reflect shorter wavelength light while allowing longer wavelength light to pass through. Therefore, the excitation light will be reflected to the specimen to pass through the objective lens and then excite the fluorophore. The emitted photons are red-shifted by about 20 nm and will pass through the dichroic mirror 750. To improve the signal-to-noise ratio, an emission filter 730 is applied to block background light of other wavelengths. Finally, the emitted light passes through the eyepiece 760 for better magnification and focusing.

[0083] The standard method of fluorescence imaging used in fluorescence microscopy is described above. FIG. 13( b) presents a simplified alternative design of system 800 that is more portable and compatible with smartphone cameras. System 800 uses only one emission filter 830 and one green laser (532 nm) 810. In the embodiment shown, the green laser 810 is used as a coherent light source and is angled at 45° relative to the chip surface. The DNA microarray can be clearly seen through the emission filter 830. Alternative light sources (e.g., light emitting diodes (LEDs) or arc lamps) can be used instead of lasers, but additional excitation filters are required and focusing is required.

[0084] Referring now back to FIG. 13( a ), an optical lens 760 is used to provide magnification. Either a scientific-grade camera or a mobile phone camera can produce clear images of the microarray for downstream image processing by specialized software. The image shown in FIG. 2( e ) was taken using a scientific-grade camera with a standard filter set. The image in FIG. 4( c ) was taken using an iPhone 6s using the settings shown in FIG. 12( b ). Alternatively, an appropriately positioned array of photodetectors may also serve to provide relevant information about the brightness of the microarray spots.

[0085] Regarding the implementation methods including the above embodiments, the following technical solutions are also disclosed:

[0086] Scheme 1. A device for multiplex amplification and detection of nucleic acid targets in a sample, the device comprising:

[0087] a mechanical system configured to provide loading, vertical positioning and clamping of the chip;

[0088] a thermal control system configured to maintain a first temperature of the chip and a second temperature of the chip, wherein the first temperature is different from the second temperature;

[0089] An optical fluorescence imaging system configured to collect at least 40 pixels

[0090] ×40 pixels in an array of spatial information;

[0091] an electrical system configured to provide electrical energy to the mechanical system, the thermal control system, and the optical fluorescence imaging system;

[0092] a controller configured to control operation of the mechanical system, the thermal control system, the power system, and the optical imaging system; and

[0093] A graphical user interface (GUI) is configured to allow a user to operate the control system via user interface software.

[0094] Solution 2. A device according to Solution 1 or 2, wherein the chip has a width of 10 mm and a width of 320 mm.

[0095] mm in height, 10 mm in width and 320 mm in width.

[0096] Thickness between mm.

[0097] Option 3. A device according to any of the preceding options, wherein the GUI is a touch screen interface.

[0098] Option 4. An apparatus according to Option 3, wherein the touch screen interface is integrated into a smart phone.

[0099] Option 5. A device according to any of the preceding options, wherein the mechanical system includes a chip holder, a frame, a sliding part and a supporting part.

[0100] Option 6. A device according to Option 5, wherein the chip holder includes a slot, a positioning groove and a motion control kit.

[0101] Solution 7. The device according to Solution 5, wherein the frame includes a base, a main structure,

[0102] components, strut positioners, struts and motion control kits.

[0103] Solution 8. The device according to solution 7, wherein the motion control kit includes a motorized movement

[0104] Components and their retainers.

[0105] Solution 9. The device according to solution 8, wherein the motorized moving part includes a linear actuator

[0106] stepper motor.

[0107] Solution 10. The device according to Solution 5, wherein the sliding component comprises a sliding platform and a sliding

[0108] Moving rod.

[0109] Solution 11. The device according to Solution 5, wherein the support member comprises a rail bracket. Solution 12. The device according to any of the preceding solutions, wherein the thermal control system comprises

[0110] Multiple temperature sensors, multiple heating blocks and heat sources.

[0111] Embodiment 13. The device according to embodiment 12, wherein the plurality of temperature sensors include resistance temperature sensors.

[0112] Temperature detector (RTD), thermistor, thermocouple or IR sensor.

[0113] Solution 14. The device according to solution 12, wherein a temperature sensor among the plurality of temperature sensors

[0114] The temperature sensor is embedded in the heating block.

[0115] Solution 15. The device according to solution 12, wherein a temperature sensor among the plurality of temperature sensors

[0116] A temperature sensor is connected to a surface of a heating block among the plurality of heating blocks.

[0117] Option 16. A device according to Option 12, wherein the plurality of heating blocks comprise aluminum, stainless steel

[0118] Or brass.

[0119] Option 17. The device according to Option 12, wherein the plurality of heating blocks contact the chamber together.

[0120] At least 50% of the total surface area.

[0121] Option 18. The device according to Option 12, wherein the heat source comprises a bonded flexible heater,

[0122] Heat probe or heating wire.

[0123] Option 19. A device according to any of the preceding options, wherein the fluorescence imaging system comprises a light source, an optical module, and a detector having at least 40×40 pixels.

[0124] Option 20. The device of Option 19, wherein the detector is a camera.

[0125] Solution 21. The device according to Solution 20, wherein the camera comprises a scientific grade camera or an intelligent

[0126] Phone camera.

[0127] Solution 22. The device according to solution 19, wherein the light source comprises an arc lamp, a vapor lamp,

[0128] Light Emitting Diode (LED) or Laser.

[0129] Option 23. The device according to Option 19, wherein the optical module comprises an excitation filter,

[0130] A dichroic mirror, a beam splitter, an emission filter, a plane mirror, an objective lens and / or an optical lens. Solution 24. A device according to any of the preceding solutions, wherein the mechanical system comprises a

[0131] One or more plastics.

[0132] Embodiment 25. A device according to any of the preceding embodiments, wherein the mechanical system comprises a polymer

[0133] Lactic acid (PLA), polycarbonate (PC), acetonitrile butadiene styrene (ABS) or ceramic. Solution 26. The device according to any of the preceding solutions, wherein the power system includes an AC / DC power supply, a MOSFET, a switch, an amplifier, a diode, a transistor and a resistor.

[0134] Solution 27. A device according to any one of the preceding solutions, wherein the controller comprises a microcontroller

[0135] controller and / or PID controller.

[0136] Option 28. A device according to Option 27, wherein the microcontroller comprises a Raspberry Pi, an Arduino or a Genuino.

[0137] Scheme 29. A method for analyzing a sample in a chip using the device according to Scheme 1

[0138] The method comprises:

[0139] loading a chip into the device;

[0140] operating the mechanical system to clamp the chip between a first heating block and a second heating block;

[0141] heating the first heating block to a first temperature;

[0142] heating the second heating block to a second temperature, wherein the second temperature is different from the first temperature;

[0143] directing excitation light from a light source to a surface of the chip;

[0144] detecting emitted light from the chip; and

[0145] The emitted light from the chip is analyzed.

[0146] Embodiment 30. The method according to embodiment 29 further comprises generating a data report, wherein the data

[0147] An analysis of the light emitted from the chip is reported.

[0148] 31. The method according to claim 29, further comprising:

[0149] Release the chip between the second heating blocks.

[0150] Option 32. The method according to Option 29 further comprises unloading the chip from the device.

[0152] Option 33. A method according to Option 29, wherein loading the chip includes operating a motor to perform linear movement.

[0153] Option 34. A method according to Option 29, wherein loading the chip includes operating a mechanism configured to pull the chip into the device.

[0154] Option 35. A method according to Option 29, wherein clamping the chip includes operating a mechanism using a self-locking motor, a cam follower combination or a spring.

[0155] Option 36. A method according to Option 29, wherein during operation, the first temperature is maintained between 75°C and 105°C.

[0156] Option 37. A method according to Option 29, wherein during operation, the second temperature is maintained between 30°C and 75°C.

[0157] Embodiment 38. A method according to Embodiment 29, wherein the first temperature and the second temperature are controlled by a microcontroller program, and the microcontroller program changes the heat source power based on feedback from a temperature sensor.

[0158] Embodiment 39. The method of embodiment 29, wherein the first temperature and the second temperature are controlled by a proportional-integral-derivative (PID) controller.

[0159] Option 40. A method according to Option 29, wherein a first temperature sensor is embedded in the first heating block.

[0160] Option 41. A method according to Option 29, wherein a second temperature sensor is embedded in the second heating block.

[0161] Option 42. A method according to Option 29, wherein a first temperature sensor is connected to the surface of the first heating block.

[0162] Option 43. A method according to Option 42, wherein a second temperature sensor is connected to the surface of the second heating block.

[0163] Option 44. A method according to Option 29, wherein the excitation light source forms an angle between 30° and 90° with the surface of the chip.

[0164] Option 45. A method according to Option 29, wherein detecting the emitted light from the chip includes operating a camera to continuously acquire images at a frequency of not less than 1 image every 2 minutes.

[0165] In view of the present disclosure, all methods disclosed and claimed herein can be performed and executed without undue experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods described herein and the steps of the methods or the order of the steps without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can replace the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0166] V. References

[0167] The following references are expressly incorporated herein by reference to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

[0168] PCT Patent Publication WO 2017 / 172760

Claims

1. A device for multiplex amplification and detection of nucleic acid targets in a sample, the device comprising: a mechanical system configured to provide loading, vertical positioning and clamping of the chip; a thermal control system configured to maintain a first temperature of the chip and a second temperature of the chip, wherein the first temperature is different from the second temperature; an optical fluorescence imaging system configured to collect spatial information in an array of at least 40 pixels by 40 pixels; an electrical system configured to provide electrical energy to the mechanical system, the thermal control system, and the optical fluorescence imaging system; a controller configured to control operation of the mechanical system, the thermal control system, the power system, and the optical imaging system; as well as A graphical user interface (GUI) is configured to allow a user to operate the control system via user interface software.

2. The device according to claim 1 or 2, wherein the chip has a height between 10 mm and 320 mm, a width between 10 mm and 320 mm and a thickness between 0.5 mm and 10 mm.

3. The apparatus of any preceding claim, wherein the GUI is a touch screen interface. The device of claim 3 , wherein the touch screen interface is integrated into a smartphone.

5. The device according to any of the preceding claims, wherein the mechanical system comprises a chip holder, a frame, a sliding part and a supporting part.

6. The device of claim 5, wherein the chip holder comprises a slot, a positioning groove and a motion control kit.

7. The apparatus of claim 5, wherein the frame comprises a base, a primary structural carrier, a strut positioner, a strut, and a motion control kit.

8. The device according to claim 7, wherein the motion control kit comprises a motorized moving part and a holder thereof.

9. The device of claim 8, wherein the motorized moving member comprises a linear actuator or a stepper motor.

10. The device according to claim 5, wherein the sliding component comprises a sliding platform and a sliding rod.

Images